This invention relates to methods and apparatus for measuring an oxygen concentration gradients and method of use thereof, and computer program products therefor.
Although others have attempted to measure oxygen concentrations to provide information regarding tissues and other in vivo environments, e.g., Vanderkooi et al., J. Biol. Chem., 262 (12):5476-5482 (April 1987); U.S. Pat. No. 4,476,870; U.S. Pat. No. 4,947,850; U.S. Pat. No. 5,186,173; U.S. Pat. No. 5,515,864, there has remained a need in the art for an apparatus and system that will quickly, accurately, and economically measure oxygen concentrations throughout a sample permitting calculation of the oxygen concentration gradient as a reliable and reproducible characterization or diagnostic tool.
The present inventors have responded to the need for an improved, reliable and fast way of measuring the oxygen concentration gradient in a sample by developing an novel apparatus and accompanying method of calculating linear oxygen concentrations in the sample, permitting diagnostic testing, for example, of the effects of a developmental or metabolic change in a cell or tissue, in vitro or in vivo, in response to disease, injury, radiation, or mechanical or chemical intervention, or simply to changed circumstances, or to measure the oxygen permeability of a membrane or plastic.
In accordance with one aspect of the present invention, there is provided an apparatus comprising:
a core digital signal processor (DSP), having sufficient memory (RAM and ROM) to perform the necessary calculations, to control output of the excitation source, and to collect phosphorescent lifetime data;
a first Delta Sigma signal processor (D/A, digital to analog) for converting tabulated calculated data to current to control an excitation light signal from the selected light source;
an avalanche photodiode to photomultiplier for filtering and detecting emitted phosphorescent light from the sample following exposure to the excitation light signal;
an amplifier for amplifying the output of the photodiode or photomultiplier; and
a second Delta-Sigma signal processor (A/D analog to digital) responsive to the amplified output from the photodiode or photomultiplier, for digitizing the amplified photodetector output (the emitted phosphorescence), and for compiling collected data into a separate memory set, m (the tabulated calculated data), in the DSP, wherein data is summed to recover distribution of the phosphorescent lifetimes, from which oxygen concentration gradient is calculated from at least one equation.
In another embodiment of the invention an apparatus is provided, wherein the data collected by the second signal processor (the digitizer) is synchronized with the first signal processor (the D/A unit) to control the driving current controlling the selected light source. The preferred apparatus relies on the principle that the emitted phosphorescence is functionally related to oxygen quenching when exposed to excitation light, and that the light source introduces a plurality of signals into the sample, such that a set of signals is established in the sample, wherein a waveform is derived, and wherein all component waveforms pass through zero.
An apparatus is also provided, wherein the photodetector or photomultiplier detects a plurality or emitted signals corresponding to a plurality or excitation signals introduced into the sample as the excitation light, and wherein the detection means determines a solution of at least one equation based upon variations in the respective values of the signal parameters of the plurality of detected emission signals. In a preferred embodiment all modulation frequencies are mixed in a excitation light, the oxygen concentration gradient is extracted from a dependence of phosphorescence amplitude and phase angle on the modulation frequency in the plurality of detected signals.
An apparatus is further provided, wherein the photodetector or photomultiplier detects a plurality of emitted signals corresponding to a plurality of emitted signals (phosphorescence), wherein the frequency and amplitude of said emitted signals is inversely related to quenching by oxygen in the sample, and wherein the detection means determines a solution of at least one equation based upon variations in the respective values of the signal parameters of the plurality of detected emission signals.
The invention further provides an apparatus, wherein the detection signal processor further comprises a means for regularizing the detected phosphorescence signals; and a means, responsive to said regularizing means, for representing the regularized signals by a Maximum Entropy solution using fast, non-iterative quadratic programming algorithm at each maximizing step to interpolate a histogram representing the best underlying distribution of the phosphorescence lifetimes. In addition, the preferred apparatus further converts the histogram representing the best underlying distribution of phosphorescence lifetimes into a distribution of oxygen concentrations by the Stern-Volmer relationship.
In certain additional embodiments of the invention, the apparatus further comprises a high sensitivity video camera for measuring the emitted phosphorescence from the phosphorescent compound. One or more steps of the method or apparatus may also be automated.
Further provided, is a method for determining an oxygen concentration gradient in a sample comprising: (i) dissolving or introducing a hydrophilic phosphorescent compound in the sample, wherein quenching constant and lifetime at zero oxygen are known or previously determined for the phosphorescent compound; (ii) illuminating the sample with a pulsed or modulated excitation light at an intensity and frequency sufficient to cause the phosphorescent compound to emit a measurable phosphorescence; (iii) measuring the emitted phosphorescence; and (iv) calculating the phosphorescence lifetime and oxygen concentration gradient in the sample.
The invention also provides a computer program product for determining oxygen concentration gradient from detected phosphorescence lifetimes in a phosphor-containing sample based upon a signal that has propagated through at least a portion of the sample, wherein the signal varies with respect to excitation frequencies from an excitation light source and emitted phosphorescence, wherein the emitted phosphorescence varies in an inverse direct relationship to oxygen quenching in the sample, and wherein the computer program product comprises a computer-readable storage medium having computer-readable program code means embodied in said medium, said computer-readable program code means comprising:
a first computer-readable program code means for analyzing the emitted phosphorescence signal detected from the sample to determine variations in the signal with respect to a predetermined quenching constant and maximal lifetime at zero oxygen for the phosphor;
a second computer-readable program code means, responsive to said first computer-readable program code means, for constructing one or more equations at least partially based upon the signal, wherein an equation extracts the dependence of phosphorescence amplitude and phase angle with the summation of modulation frequencies in the excitation light;
a third computer-readable program code means, responsive to the second computer-readable program code means, for determining a solution of the one or more equations, which has been constructed to resolve the variations in phosphorescence amplitude and phase angle with respect to modulation frequencies and the quenching constant and maximal lifetime at zero oxygen for the selected phosphor;
a fourth computer-readable program code means, responsive to the third computer-readable program code means, for determining the solution of the one or more equations, wherein the fourth computer-readable program means comprises computer-readable program code means for recovering an algorithmically-determined histogram which maximally resembles the phosphorescence lifetime distribution of the selected phosphor in the sample; and
a fifth computer-readable program code means, responsive to the fourth computer-readable program code means, for determining the solution of the one or more equations, wherein the fifth computer-readable program means comprises computer-readable program code means for algorithmically-converting the phosphorescence lifetime distribution into a corresponding oxygen concentration gradient based upon the Sterne-Volmer relationship.
In addition, the present invention provides methods of using the apparatus described above to detect phosphorescence lifetimes in a phosphor-containing sample, and in preferred embodiments to determine therefrom an oxygen concentration gradient in a phosphor-containing sample.